Dopamine Beta-hydroxylase (DBH) catalyzes the synthesis of the neurotransmitter noradrenaline in storage vesicles of the neuron (synaptic vesicles) or the adrenal medulla (chromaffin granules). The subunit structure of the 280,000 molecular weight native protein complex will be determined. The results from these studies will form the basis for the determination of the complete amino acid sequence of the DBH polypeptide chain which will be achieved using classical techniques of peptide fragmentation, column chromatography, amino acid analysis and sequence analysis modified for high sensitivity. It will then be explored whether the mode of subunit interaction is via inter-chain disulfide bonds. Disulfide-containing peptides, representing both the inter- and the intra-subunit disulfide links, will be isolated from peptic digests of native DBH and characterized, thus completing the determination of the complete covalent structure of DBH. Chemical modification of critical amino acids will be performed and modified peptides isolated in order to illuminate structure-activity relationships and molecular topography of DBH. Similar peptide mapping techniques will be used to determine whether the polypeptide chain of chromogranin A is related to DBH. The interaction of endogenous inhibitors with DBH will be examined by biochemical and spectral techniques. The topography of DBH and the other chromaffin granule proteins on the limiting membrane will be characterized using radioactive labeling and enzymatic procedures. The importance of lipids to DBH activity will be studied using free lipids and liposomes in order to yield further information on the nature of the interaction of DBH with its subcellular environment. BIBLIOGRAPHIC REFERENCES: R.A. Hogue-Angeletti, "Characterization of Chromaffin Granule Components," Abstr. Amer. Soc. Neurochemistry, (1976) 240. W. Lysiak and R.A. Hogue-Angeletti, "A Serum Inhibitor of Dopamine Beta-Hydroxylase," Fed. Proc. Abstr., 1977.